Inositol phosphatase SHIP1 is a primary target of miR-155

Abstract

MicroRNA-155 (miR-155) has emerged as a critical regulator of immune cell development, function, and disease. However, the mechanistic basis for its impact on the hematopoietic system remains largely unresolved. Because miRNAs function by repressing specific mRNAs through direct 3'UTR interactions, we have searched for targets of miR-155 implicated in the regulation of hematopoiesis. In the present study, we identify Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1) as a direct target of miR-155, and, using gain and loss of function approaches, show that miR-155 represses SHIP1 through direct 3'UTR interactions that have been highly conserved throughout evolution. Repression of endogenous SHIP1 by miR-155 occurred following sustained over-expression of miR-155 in hematopoietic cells both in vitro and in vivo, and resulted in increased activation of the kinase Akt during the cellular response to LPS. Furthermore, SHIP1 was also repressed by physiologically regulated miR-155, which was observed in LPS-treated WT versus miR-155(-/-) primary macrophages. In mice, specific knockdown of SHIP1 in the hematopoietic system following retroviral delivery of a miR-155-formatted siRNA against SHIP1 resulted in a myeloproliferative disorder, with striking similarities to that observed in miR-155-expressing mice. Our study unveils a molecular link between miR-155 and SHIP1 and provides evidence that repression of SHIP1 is an important component of miR-155 biology.

Fig. 1.

MicroRNA-155 represses SHIP1 expression through 3′UTR interactions. (A) Schematic layout of the SHIP1 mRNA coding sequence (CDS) and 3′UTR, with the relative location of the miR-155 binding site. Depiction is not to scale. Sequence of mouse and human miR-155 and predicted interaction with conserved 8-mer miR-155 seeds found within the SHIP1 3′UTRs from different species (highlighted) are shown. The sequence of the SHIP1 3′UTR seed mutant used for reporter assays and predicted disruption of the miR-155 interaction is also shown. (B) Luciferase reporter assays were performed by transiently transfecting 293T cells with an empty plasmid (FUW) or miR-155-expressing plasmid (FUW-155), the indicated 3′UTR luciferase reporter plasmids, and a plasmid producing β-galactosidase. Luciferase values have been normalized to β-galactosidase, and the percent of luciferase activity in cells transfected with miR-155 is presented. Raw 264.7 cells stably infected with a retroviral vector expressing WT human miR-155 (MGP-h155), mutant seed human miR-155 (MGP-h155mut), or control (MGP) were assayed for SHIP1 levels by qPCR (C) and Western blotting (D). As a loading control for the Western blot, α-Tubulin was also assayed. The fold-repression of SHIP1 by the different constructs is shown. (E) Levels of mature human miR-155 in the different cell types were assayed by qPCR with primers that detect the WT mature miR-155 sequence. (F) RNA from the different cell types and a probe specific for the human miR-155 seed mutant sequence was used for Northern blotting. (G) The different cell types were stimulated with LPS (200 ng/ml) over the indicated time course and Ser-473-phosphorylated-Akt (p-Akt), total Akt, and β-Actin were assayed by Western blotting. Data represent at least 2 independent experiments.

Fig. 2.

Enhanced expression of SHIP1 in miR-155^−/− macrophages following LPS treatment. Bone marrow-derived macrophages from WT or miR-155^−/− mice were stimulated with 10-ng/ml LPS from Escherichia coli for the indicated periods of time. Expression of BIC (A) or mature miR-155 (B) was assayed by qPCR. Expression of SHIP1 was assayed in BMMs by Western blotting (C) and qPCR (D). β-Actin was assayed as a loading control for the Western blot, while qPCR data were normalized to L32. The fold-increase in SHIP1 expression versus the WT 0-h sample is shown. Data represent at least 2 independent experiments.

Fig. 3.

Knockdown of SHIP1 in vivo using siRNA in the context of miR-155 processing. (A) Schematic of the retroviral vector (MGP-155f) used to deliver siRNA against SHIP1 in miR-155 format. (B) Knockdown of SHIP1 was assayed in Raw 264.7 cells infected with MGP-siSHIP1 or control vector by Western blotting. α-Tubulin was assayed as a loading control. (C) Knockdown of SHIP1 in vivo by retroviral expression of miR-155 (MGP-155, n = 4 mice) or siSHIP1 (MGP-siSHIP1, n = 3 mice) in the hematopoietic compartment was assayed by qPCR using RNA isolated from total bone marrow following 2 months of hematopoietic reconstitution. Relative expression values have been normalized to L32 mRNA. A P-value of 0.05 or less using a Student's t test was considered statistically significant and indicated with an asterisk.

Fig. 4.

Knockdown of SHIP1 or expression of miR-155 in the hematopoietic compartment cause similar MPDs in the bone marrow. (A) Bone marrow was extracted from mice expressing human miR-155 (MG-155, n = 3 mice), mouse miR-155 (MGP-155, n = 4 mice), siSHIP1 (MGP-siSHIP1, n = 3 mice), or control vectors (MG, n = 2 mice or MGP, n = 3 mice) 2 months following bone marrow reconstitution. Total bone marrow cells were assayed for expression of CD11b (Mac1), Ter119, or B220 using flow cytometry. Each dot represents an individual mouse. A P-value of 0.05 or less using a Student's t test was considered statistically significant and indicated with an asterisk. (B) Bone marrow from MGP, MGP-155, or MGP-siSHIP1 mice was smeared and Wright-stained. Photomicrographs are shown (1,000× magnification). (Scale bar, 20 μm.) (C) Representative flow cytometry plots from control, MGP-155 and siSHIP1 vector-containing mouse bone marrow analyzing GFP and CD11b expression.

Fig. 5.

Knockdown of SHIP1 or expression of miR-155 in the hematopoietic compartment causes splenomegaly and extramedullary hematopoiesis in the spleen. (A) Spleens were extracted from mice expressing human miR-155 (MG-155, n = 3 mice), mouse miR-155 (MGP-155, n = 4 mice), siSHIP1 (MGP-siSHIP1, n = 3 mice), or control vectors (MG, n = 2 or MGP, n = 3 mice) 2 months following bone marrow reconstitution. Spleens were weighed and RBC-depleted splenocytes subsequently assayed for expression of CD11b, Ter119, or B220 by FACS. Each dot represents an individual mouse. A P-value of 0.05 or less using a Student's t test was considered statistically significant and indicated with an asterisk. (B) Spleens from MGP, MGP-155, or MGP-siSHIP1 mice were fixed, sectioned, and H&E stained. Photomicrographs are shown (400× magnification). (Scale bar, 50 μm.)